Noninvasive Visualization of Respiratory Viral Infection Using

ARTICLE

Noninvasive Visualization of Respiratory Viral Infection Using Bioorthogonal Conjugated NearInfrared-Emitting Quantum Dots Hong Pan,†,§ Pengfei Zhang,†,§ Duyang Gao,† Yijuan Zhang,† Ping Li,† Lanlan Liu,† Ce Wang,† Hanzhong Wang,‡ Yifan Ma,†,* and Lintao Cai†,* †

Guangdong Key Laboratory of Nanomedicine, CAS Key Laboratory of Health Informatics, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, People's Republic of China, and ‡State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, People's Republic of China. §These authors contributed equally to this paper.

ABSTRACT Highly pathogenic avian influenza A viruses are emerging pandemic

threats in human beings. Monitoring the in vivo dynamics of avian influenza viruses is extremely important for understanding viral pathogenesis and developing antiviral drugs. Although a number of technologies have been applied for tracking viral infection in vivo, most of them are laborious with unsatisfactory detection sensitivity. Herein we labeled avian influenza H5N1 pseudotype virus (H5N1p) with near-infrared (NIR)-emitting QDs by bioorthogonal chemistry. The conjugation of QDs onto H5N1p was highly efficient with superior stability both in vitro and in vivo. Furthermore, QD-labeled H5N1p (QD-H5N1p) demonstrated bright and sustained fluorescent signals in mouse lung tissues, allowing us to visualize respiratory viral infection in a noninvasive and real-time manner. The fluorescence signals of QD-H5N1p in lung were correlated with the severity of virus infection and significantly attenuated by antiviral agents, such as oseltamivir carboxylate and mouse antiserum against H5N1p. The biodistribution of QD-H5N1p in lungs and other organs could be easily quantified by measuring fluorescent signals and cadmium concentration of virus-conjugated QDs in tissues. Hence, virus labeling with NIR QDs provides a simple, reliable, and quantitative strategy for tracking respiratory viral infection and for antiviral drug screening. KEYWORDS: bioorthogonal chemistry . near-infrared quantum dots . live virus labeling . viral infection . in vivo dynamic imaging

H

ighly pathogenic avian influenza A viruses are emerging pandemic threats that have caused several outbreaks with high mortality in human beings in the past decades.1 The infection of avian influenza viruses, such as subtype H5 and H7, could cause a series of severe respiratory and extra-respiratory complications, both of which are directly associated with viral loading and dissemination in tissues.2 Hence, understanding the route and pathogenesis of avian influenza viral infection is extremely important for developing prophylactic and therapeutic strategies against viral infections. Although the studies using cell cultures have made a significant contribution to exploring the pathogenesis of avian influenza viruses, it is highly desirable to track the in vivo dynamics of viral infection in order to further PAN ET AL.

dissect the interactions between the virus and the host.3,4 The integration of reporter genes, such as fluorescent proteins and luciferase, into viral genomes has been reported as a promising approach for noninvasive virus tracking in vivo.5,6 However, developing genetically engineered viruses requires special expertise and is timeconsuming. In addition, the fluorescent imaging of such viruses relies on reporter gene expression, which might limit the detection sensitivity, especially during the early stage of viral infection.6,7 Virus labeling with organic fluorescent dyes has been recently demonstrated as a promising alternative to track viral invasion in vitro.7 However, the instability and photobleaching of organic dyes potentially limit their application for monitoring virus infection in vivo.8 VOL. 8

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* Address correspondence to [email protected], [email protected]. Received for review September 28, 2013 and accepted May 5, 2014. Published online May 05, 2014 10.1021/nn501028b C 2014 American Chemical Society

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cadmium (Cd) concentration of virus-conjugated QDs using inductively coupled plasma/optical emission spectrometry (ICP-OES). RESULTS AND DISCUSSION Labeling of H5N1p with NIR QDs via Bioorthogonal Chemistry. In the present study, CdSeTe/ZnS alloyed core/shell structured NIR QDs were fabricated using a hot colloidal synthesis approach as previously described.31,32 The native hydrophobic ligands of QDs were then replaced with multidentate polymer ligands bearing imidazole pendant groups (N3-PMAH) through ligand exchange to obtain water-dispersible azido-derivatized NIR QD (N3-QDs) (Supporting Information Figure S1a). The average diameter of N3-QDs measured by transmission electron microscopy (TEM) was 5.7 nm (see Figure S1b), and the hydrodynamic diameter of the N3-QDs was ∼13.0 nm (Figure S1c). The N3-QDs had emission wavelength at 750 nm (Figure S1d), and the quantum yield of as-prepared QDs was about 40% (see Methods in the Supporting Information). The results of the MTT assay showed that 1 to 200 μg/mL of N3-QDs did not significantly affect the viabilities of human lung epithelial A549 cells, murine RAW 264.7 macrophages, and HEK293T cells after 24 h incubation, suggesting the cytotoxicity of N3-QDs was minimal (Figure S1e). Bioorthogonal chemistry, which can be conducted under mild conditions, has been reported as an effective method for live virus labeling with QDs.27,28 We previously established bioorthogonal chemistry for live virus labeling that allowed the specific QD labeling of the viral particle by cycloaddition between the azide moieties on the N3-QDs and the dibenzocyclooctyl (DBCO) tag on the virus.27 In the present study, the purified H5N1p was first reacted with DBCO-PEG4-NHS ester so that DBCO could be effectively conjugated onto the amine group of virus surface proteins. Azidelabeled QDs were then selectively reacted with DBCOmodified H5N1p (DBCO-H5N1p) through the bioorthogonal chemistry to acquire QD-labeled H5N1p (QD-H5N1p) (Figure 1a,c). The hydrodynamic sizes of H5N1p and QD-H5N1p were 100 ( 7.8 and 122 ( 9.7 nm, respectively (Figure 1c). The slight increase of the hydrodynamic size indicated a successful QD conjugation onto the viral particles. Moreover, the bioorthogonal conjugation onto the viral particles did not significantly affect fluorescence intensity or emission spectrum of QDs (Figure 1d). To determine the optimal dose of N3-QDs, H5N1p was reacted with different amounts of N3-QDs at ratios of H5N1p to N3-QDs from 2.5 to 40 (w/w). The virusbound and unbound QDs could be separated by the agarose gel electrophoresis due to their different particle sizes.33 The results showed that when the ratio of H5N1p to N3-QDs was 20 or above, unbound N3-QDs were not detectable (Figure 1b), suggesting that QDs were effectively conjugated onto H5N1p. Therefore, VOL. 8

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In the past decade, viral particle labeling with quantum dots (QDs) has emerged as an effective strategy for virus tracking. Compared with conventional fluorescent dyes, QDs demonstrate bright photoluminescence, broad size-tunable emission spectrum, and photochemical stability and have been successfully applied for single-virus tracking in vitro.9
13 Liu et al. developed a dual-labeling strategy for single-virus imaging, which labeled the viral envelope and genome with QDs and Syto 82, respectively, and allowed monitoring of both viral particle invasion and viral genome transportation by host cells.14 Joo et al. showed that conjugating QDs to AAV (adeno-associated virus) capsid via the carbodiimide chemistry did not affect either the infectivity or the intracellular transport properties of the virus.15 Unfortunately, it remains a big challenge to use QDs to track viral infection in vivo. An ideal fluorescent labeling for in vivo virus tracking must be bright, stable, and capable of penetrating tissues. Recently, QDs with near-infrared (NIR) spectrum from 700 to 900 nm have attracted intense attention due to the ability of deep-tissue imaging in this wavelength range.8,16
19 NIR QDs have been successfully applied for noninvasive tumor molecular imaging and diagnosis,20 in vivo drug distribution monitoring,21,22 and lymphatic system imaging.23 Their application for visualizing viral infection in vivo, especially in deep tissues and organs, has not been well-established. Moreover, the stability of QD conjugation is also a critical issue for tracking viral infection in vivo. Bioorthogonal chemistry, a biocompatible chemical reaction that can occur in the presence of other rich chemical functionalities found in biological systems without interacting or interfering with native biochemical processes, allowed a wide range of bioactive molecules to be specifically labeled in living organisms.24
26 Recent studies reported that the copper-free click chemistry was a simple, effective, and bioorthogonal strategy for live viral labeling and did not significantly affect viral infectivity.27,28 Avian influenza H5N1 pseudotype virus (H5N1p) has been reported as a safe surrogate of H5N1 virus with similar endocytic pathways and pathological changes as wild-type virus.29,30 Herein we labeled avian influenza H5N1p with NIR QDs by bioorthogonal reaction27 and dynamically monitored the respiratory infection of QD-labeled H5N1p (QD-H5N1p) in mice using an in vivo imaging system. QD-H5N1p demonstrated bright and sustained fluorescent signals in mouse lung tissues, which were strongly correlated with the severity of viral infection that was verified by lung histology. We also explored the potential application of QD-H5N1p for antiviral drug evaluation. The results showed that the administration of the antiviral agents oseltamivir carboxylate and mouse antiserum significantly affected the in vivo dynamics of QD-H5N1p infection, which could be directly quantified by measuring fluorescent signals and

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ARTICLE Figure 1. Bioorthogonal labeling of H5N1p with NIR QDs. (a) Schematic illustration of virus labeling with NIR QDs. (b) Agarose gel electrophoresis analysis of H5N1p reacted with different amounts of QDs. The top and bottom panels are the fluorescence image of NIR QDs and Coomassie blue staining of viral particles, respectively. (c) Particle sizes of QDs, H5N1p, and QD-labeled H5N1p (QD-H5N1p). (d) Emission spectrum and fluorescence intensity of QDs and QD-H5N1p. (e,f) Infectivity of H5N1p with or without QD conjugation. HEK293T cells were infected with H5N1p, DBCO-H5N1p, or QD-H5N1p for 24 h, and virus infection was identified by GFPþ cells using fluorescence microscopy (e). The percentages of GFPþ cells were quantified by flow cytometry (f). Bars shown are mean ( SE (n = 3
4). Scale bar = 20 μm.

the ratio of H5N1p to N3-QDs at 20 was applied for future experiments. The infectivity of DBCO-modified H5N1p (DBCO-H5N1p) and QD-H5N1p was evaluated by measuring viral GFP expression in HEK293T cells (Figure 1e). The results showed that neither DBCO modification nor QD conjugation significantly affected viral GFP expression at 24 h post-infection, suggesting that the infectivity of H5N1p was not impaired (Figure 1e,f). Hence, QD labeling with bioorthogonal chemistry is a mild and reliable strategy for live virus labeling. Stability and Infectivity of QD-H5N1p in A549 Cells. Although QDs have been successfully applied for single-virus tracking in host cells, the stability of QD conjugation remains a big concern. We therefore evaluated the intracellular stability of QD-H5N1p in human lung epithelial A549 cells. A549 cells were incubated with QDs, H5N1p, or QD-H5N1p for 3 h, and the intracellular H5N1p was identified by immunofluorescent staining with the antibody against hemagglutinin (HA), a surface glycoprotein of H5N1. The fluorescent signals of QDs were barely detectable in A549 cells after 3 h incubation with QDs alone, suggesting that the nonspecific internalization of QDs by PAN ET AL.

host cells was minimal. In contrast, both H5N1p and QD-H5N1p were effectively internalized by A549 cells and predominantly distributed in the cytosol at 3 h post-infection, suggesting that QD labeling did not affect either the internalization or intracellular localization of viral particles in A549 cells. More importantly, the green fluorescence of HA was completely colocalized with red fluorescence of NIR QDs, indicating that the viral labeling with QDs by bioorthogonal chemistry was highly efficient. The spectrum of these colocalized dots showed two separated emission peaks at 550 and 750 nm, which could be attributable to the fluorescent dye (Alexa 488) of anti-HA antibody and QDs, respectively (Figure 2a). Hence, the conjugation of QDs to H5N1p was stable in A549 cells upon virus infection (Figure 2a). Previous studies showed that QDs might not be stable in intracellular compartments, such as lysosomes and peroxisomes.34 The intracellular biothiols (e.g., glutathione and cysteine) have been shown to degrade the monolayer of QDs, thereby causing QD aggregation and loss of fluorescence.35 In the present study, H5N1p-conjugated NIR QDs remained detectable with an unshifted fluorescence emission spectrum VOL. 8

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ARTICLE Figure 2. Intracellular localization of QD-H5N1p in A549 cells. A549 cells were treated with H5N1p, QDs, or QD-H5N1p for 3
24 h. (a) Three hours after infection, the cells were labeled with anti-HA antibody, followed by Alexa 488-conjugated antiIgG (green) to identify viral particles. The fluorescent images of the cells and the spectrum of pointed fluorescent dots were recorded by the multispectral imaging system. Scale bar = 5 μm. (b) Fluorescent images of H5N1p or QD-H5N1p-infected A549 cells and the spectrum of pointed fluorescent dots at 24 h post-infection. Scale bar = 5 μm. Red lines: the fluorescent spectrum.

in virus-infected A549 cells at 24 h after virus infection, indicating their excellent intracellular stability (Figure 2b). The pretreatment of chloroquine (a lysosome inhibitor) did not affect either fluorescence intensity or emission spectrum of virus-conjugated NIR QDs (Figure S2), further suggesting their stability in endosomes and lysosomes. The long-term intracellular stability of QDs might be due to the imidazole-based multidentate polymer ligands of a QD monolayer, which were reported to enhance colloidal stability of nanocrystals more effectively than thiol-based monodendate ligands.35
37 The bioorthogonal conjugation of NIR QDs to viral particles was highly efficient with remarkable stability in virus-infected cells, which would allow us to monitor the long-term dynamics of viral infection. In Vivo Dynamics of Respiratory Viral Infection in Mice. To study the in vivo dynamics of avian influenza virus infection, Balb/c mice were intranansally (i.n.) infected with live or heat-inactivated QD-H5N1p for 12
72 h, and the fluorescence signal of QDs in lungs was recorded using an in vivo imaging system. For comparison, some mice inhaled equal amounts of QDs. The inhalation of QDs resulted in dim and transit fluorescent signals in lungs, which primarily accumulated in the PAN ET AL.

trachea, bronchii, and upper lobes (Figure 3a
c). In contrast, the infection of live QD-H5N1p resulted in bright and sustained fluorescent signals in lungs from 12 to 72 h post-infection with the maximum fluorescence intensity at 24 h. Notably, the fluorescent signals of QD-H5N1p spread to whole lung tissue at 72 h, which should be associated with virus diffusion into lower respiratory tract and pulmonary alveoli.5 Compared with live QD-H5N1p, the infection of heatinactivated QD-H5N1p led to dramatically decreased and restricted fluorescent signals in lungs, similar to free QDs did (Figure 3). These data suggested a strong correlation between fluorescent signals of QD-H5N1p and virus infectivity. To further confirm the conjugation of QDs to H5N1p in vivo, lung sections from QD-H5N1p-infected mice were labeled with anti-HA antibody to identify viral particles. Similar to the observation in vitro, the immunofluorescent imaging showed nice colocalization of QDs and H5N1 HA in lung epithelium 3 days post-infection. The fluorescence spectrum of the colocalized dots in lung tissues showed two separate emission peaks at 550 and 750 nm, which could be attributable to the fluorescent dye (Alexa 488) of VOL. 8

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ARTICLE Figure 3. In vivo imaging of respiratory QD-H5N1p infection in mice. Six week old female Balb/c mice were i.n. infected with live or heat-inactivated QD-H5N1p, or with equal amounts of QDs as described in Methods. (a,b) Fluorescent images of mice were recorded at 12
72 h post-infection. The fluorescent signals of circled areas were analyzed using the Maestro software, and the average fluorescence intensity was calculated using the following formula: average signals (counts/s/pixel) = total counts/exposure time (s)/area (pixel). Bars shown are mean ( SE (n = 3
4). (c) Fluorescence images of lungs on day 3 postinfection.

antibodies and QDs as previously observed (Figure 4a,b). These data clearly demonstrated a stable conjugation of NIR QDs to live viruses in vivo. Surprisingly, QDs were retained in viral-infected pulmonary epithelium with bright fluorescence and unshifted emission spectrum even on day 6 post-infection (Figure 4c,d). These data indicated the stability of virus-conjugated NIR QDs, which would be essential for long-term tracking of virus infection in vivo. The bioorthogonal labeling of NIR QDs could be applied to noninvasively monitor the dynamics of respiratory viral infection in live mice. It is worthy to note that, although the bioorthogonal QD labeling may not be suitable for tracing virus progeny, it allows for the analysis of the route of viral invasion in small animals, which could be important for virology research. Effect of Antiviral Agents on In Vivo Dynamics of Respiratory Viral Infection in Mice. Antiviral drugs and neutralizing antibodies are effective strategies to prevent and treat avian influenza-caused illnesses.38
40 It is well-known that neuraminidase inhibitors, such as oseltamivir and zanamivir, can prevent the spread of influenza virus by the blocking of viral budding.38 More recent studies showed that oseltamivir carboxylate (an active form of neuraminidase inhibitors) also inhibited viral entry at the early stage of viral infection.41
43 Passive immunization with neutralizing antibodies has emerged as another important approach to control influenza virus infection.44 However, their effect on in vivo dynamics of virus infection remains unclear. In the present study, PAN ET AL.

QD-H5N1p was pretreated with oseltamivir carboxylate or mouse antiserum against H5N1p. The pretreatment of oseltamivir carboxylate or antiserum did not significantly affect the fluorescence intensity of QD-H5N1p. The hydrodynamic size of QD-H5N1p was selectively increased by antiserum but not by negative serum (Figure S3). These data suggested antibodytriggered virus aggregation, which is an important mechanism for preventing virus infection.45 The infectivity of QD-H5N1p with or without pretreatment was evaluated in HEK293T cells. The results showed that the pretreatment of oseltamivir carboxylate or antiserum not only dramatically attenuated the entry of viral particles to HEK293T cells but also diminished the expression of viral GFP by ∼50 and ∼90%, respectively (Figure S4), confirming their inhibitory effect on virus entry and viral infectivity.41,45 The antiviral effect of oseltamivir carboxylate and antiserum was further investigated in vivo. The inflammatory response in lung tissues has been reported as a major mechanism contributing to H5N1-caused acute lung injury.46,47 In the present study, i.n. infection with QD-H5N1p caused significant lung inflammation, including thickening of epithelium and infiltration of inflammatory cells, on day 3 post-infection (Figure 5a,b). The QD-H5N1p significantly increased the mRNA expression of pro-inflammatory cytokines and chemokines, including IL-1β, IL-6, TNFR, and CCL2, in lung tissues (Figure S5). In contrast, the pretreatment of oseltamivir carboxylate or mouse antiserum not only VOL. 8

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ARTICLE Figure 4. Distribution of QD-H5N1p in lung tissues. (a
e) Three days after infection, lung tissue sections from QD-H5N1pinfected mice were stained with anti-HA antibody and Hoechst to identify viral particles (green) and nuclei (blue), respectively. The fluorescent imaging of lung tissues (a) and the emission spectrum of pointed fluorescent dots (b) were recorded by the multispectral imaging system. Scale bar = 100 μm. (c,d) Fluorescent images of QD-H5N1p-infected pulmonary epithelial cells (GFPþ cells) were recorded on day 6, and the spectrum of pointed fluorescent dots in the cells was measured as described previously. Scale bar = 20 μm.

attenuated lung pathology caused by virus infection but also reduced mRNA levels of proinflammatory cytokines and chemokines (Figure S5). Hence, both oseltamivir carboxylate and mouse antiserum effectively suppressed respiratory infection of QD-H5N1p in mice. The effect of antiviral agents on the dynamics of QD-H5N1p infection was further evaluated by the in vivo imaging system. Mice were i.n. infected with QD-H5N1p with or without the pretreatment of oseltamivir carboxylate or antiserum, and the fluorescent imaging of mice was recorded from 12 to 72 h postinfection. As we expected, mice infected with untreated QD-H5N1p showed the brightest fluorescence in lung tissues with the maximum fluorescence intensity at 24
48 h (Figure 5c,d). Although oseltamivir carboxylate did not significantly affect the fluorescence intensity of QD-H5N1p, the pretreatment of QD-H5N1p with oseltamivir carboxylate led to 40
60% less fluorescent signals in lungs. Furthermore, QD-H5N1p pretreated with antiserum resulted in the lowest fluorescent signals in lung tissues among three groups (Figure 5c,d). The fluorescence intensity of QDH5N1p in lungs was nicely correlated with the severity of viral infection, suggesting the bioorthogonal labeling of QDs as a promising strategy for in vivo antiviral drug screening. The ex vivo analysis of whole lungs showed that the pretreatment of antiviral agents, PAN ET AL.

especially mouse antiserum, lowers fluorescent signals of QD-H5N1p in upper lobes, which should be due to the limited virus spread in lung tissues (Figure 5e). To further determine the distribution of viral particles in lungs, lung sections were labeled with anti-HA antibody. The results showed that the untreated QDH5N1p was dispersed in the alveolar epithelium, while QD-H5N1p pretreated with antiviral agents mostly accumulated near the main conducting airways with limited diffusion into pulmonary alveoli (Figure S6). These data demonstrated the inhibitory effect of oseltamivir carboxylate and antiserum on virus diffusion in lung tissues, which further confirmed their antiviral effects. The infection of H5N1 avian influenza has been reported to cause both respiratory and extra-respiratory complications that are directly associated with viral loading and dissemination in the body.48 Therefore, understanding the biodistribution of viruses in vivo is always highly desirable for elucidating the pathology of viral infection. In the present study, since the conjugation of QDs to H5N1p via bioorthogonal chemistry demonstrated long-term stability in vivo, the distribution of QD-H5N1p in different organs could be easily quantified by measuring the tissue concentration of cadmium, the major element of NIR QDs. The results of ICP-OES showed that the infection of QD-H5N1p led to the highest amount of Cd in lung tissues among three VOL. 8

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ARTICLE Figure 5. Effect of antiviral agents on the dynamics of respiratory QD-H5N1p infection in mice. Six week old female Balb/c mice were i.n. infected by PBS or QD-H5N1p ( oseltamivir carboxylate (drug) or mouse antiserum against H5N1p (antiserum) as described in Materials and Methods. (a,b) Histopathology of lung tissues on day 3 post-infection was assessed by the H&E staining (a), followed by histopathology scoring (b). (v) Infiltrated inflammatory cells; (2) thickening of lung epithelium. Scale bar = 20 μm. (c,d) In vivo live imaging of mice at 12
72 h post-infection, and the fluorescent signals of circled regions was quantified as described in Figure 3d. (e) Ex vivo fluorescent imaging of hearts, livers, spleens, kidneys, and lungs on day 3 postinfection. (f) ICP-OES analysis of cadmium concentration in different organs 3 days post-infection. Tissue cadmium concentration (μg/g) was calculated by normalizing the amount of cadmium to sample weight. Bars shown are mean ( SE (n = 5), and the differences among groups were determined using one-way ANOVA analysis; *p < 0.05; **p < 0.01. TABLE 1. Effect of Antiviral Agents on the Biodistribution of QD-H5N1p total amount of Cd in organs (% IDa) treatment groups

heart

liver

spleen

lung

kidney

QD-H5N1p QD-H5N1p þ drug QD-H5N1p þ antiserum

0.30 ( 0.05 0.34 ( 0.08 0.49 ( 0.09

3.30 ( 1.11 5.87 ( 2.05* 5.84 ( 1.57*

0.44 ( 0.06 0.43 ( 0.09 0.43 ( 0.08

85.69 ( 14.15 54.12 ( 10.2* 53.90 ( 9.49*

1.20 ( 0.14 2.01 ( 0.25* 1.62 ( 0.39

a

Cadmium concentration in different organs was measured by ICP-OES analysis on day 3 post-infection, and the percent inoculation dose (% ID) of tissue Cd was calculated using the following formula: % ID = (Cd concentration  organ weight/inoculation dose)  100%. Data shown are mean ( SE (n = 5), and differences between QD-H5N1p and antiviral agents one-way ANOVA analysis were followed by Tukey' s post-test; *p < 0.05. PAN ET AL.

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CONCLUSION The present study reported virus labeling with bioorthogonal NIR QD conjugation as a simple and

MATERIALS AND METHODS Materials. Tellurium powder (Te, 99.8%), selenium shot (Se,99.5%), oleic acid (technical grade, 90%), tri-n-octylphosphine oxide (TOPO, 90%), zinc diethyldithiocarbamate (Zn(DDTC)2, 97%), cadmium oxide (CdO, 99.5%), and 1-octadecene (ODE, 90%) were purchased from Strem Chemicals. Oleylamine (OAm, 90%) was from Acros Organics. Poly(maleic anhydride) (MW = 5000 Da) was purchased from PolySciences, Inc., USA. Dibenzocyclooctyne (DBCO-NHS ester) was purchased from Click Chemistry Tools (Scottsdale, AZ). N3-PEG8CH2CH2NH2 (MW = 438 Da) was obtained from Biomatrik Inc., China. Unless specified, chemicals were purchased from SigmaAldrich (St. Louis, MO) and used without further purification. HEK293T and A549 cells were obtained from Shanghai cell bank of Chinese Academy of Sciences. Oseltamivir acid (ACS: 18722745-8) was purchased from Shanghai Haoyuan Chemexpress (Shanghai, China). Alexa 488-conjugated goat anti-mouse IgG antibody was from Life technology (NY, USA), and anti-HA protein (H5N1 influenza viruses) mouse monoclonal antibody (mAb) was from Sino Biological Inc. (Beijing, China). Six to eight week old Balb/c mice were obtained from Guangdong Province Laboratory Animal Center (Guangzhou, China) and maintained in the institutional animal care facility. All animal protocols were approved by Institutional Animal Care and Usage Committee of Shenzhen Institutes of Advanced Technology. Preparation and Characterization of Azido-Derivatized NIR QDs. CdTeSe/ZnS core/shell QDs (em = 750 nm) were prepared as previously reported with slight modification.31,32 Briefly, 0.56 g of oleic acid, 5 g of TOPO, and 0.26 g of Cd (OOCCH3)2 3 2H2O were heated to 100 °C and degassed for 30 min. The flask was then filled with argon gas, and its temperature was increased to 350 °C. After the precursor Cd was dissolved completely in the solvent, the temperature was decreased to 340 °C and allowed to stabilize for several minutes. At this temperature, premixed Se and Te solution, which was made by dissolving 0.64 g (1 mmol) of Te and 0.4 g in 10 mL of TOP, was quickly injected into the reaction flask. Aliquots of the reaction mixture were taken and quenched in cold chloroform. The as-prepared CdTeSe QDs were isolated through precipitation from excess acetone and then dissolved in hexane. The solution of CdTeSe QDs was diluted in a mixture of trioctylphosphine oxide (5 g) and oleylamine, and hexane was removed under vacuum at 60 °C. The solution was further evacuated for at least 30 min at 100 °C and purged with argon. After being heated to 130 °C, the ZnS shell was grown on the QDs using dropwise injection of a solution of Zn (DDTC)2. The QDs were annealed at 180 °C for 20 min to allow the growth of the ZnS monolayer. When the desired synthesis was completed, the reaction was cooled to room temperature and isolated through precipitation from excess ethanol.

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reliable strategy for tracking respiratory viral infection in vivo. The conjugation of NIR QDs was simple and stable without significantly affecting the infectivity of the virus. Furthermore, virus-conjugated QDs demonstrated bright photoluminescence and photostability and can be imaged in deep tissues, allowing long-term tracing of virus infection in a noninvasive and real-time manner. More importantly, the fluorescence signal of QD-labeled virus in lungs was strongly correlated with the severity of virus infection, and the amount of virus could be quantified by measuring both fluorescent signals and Cd concentration of virus-conjugated QDs in tissues. Hence, live virus labeling with bioorthogonal NIR QDs provides a noninvasive and quantitative tool for monitoring respiratory viral infection and antiviral drug screening.

ARTICLE

groups, while the pretreatment of oseltamivir carboxylate and antiserum dramatically reduced the concentration and total amount of Cd in lungs (Figure 5f and Table 1). These data indicated that antiviral agents significantly reduced the amount of virus loading, the major factor associated with the severity of lung infections,2 consistent with the result of lung fluorescence imaging. Interestingly, the pretreatment of oseltamivir carboxylate or antiserum increased the amount of Cd in livers and kidneys by 1
2-fold (Table 1), suggesting that the antiviral agents promoted the exit of H5N1p from lungs into blood circulation for excretion.

The as-synthesized QD750 coated with nonpolar ligands was transferred into aqueous solution by treatment with N3-PMAH, which was synthesized according to previous published work.27 The QD solution was mixed with N3-PMAH solution in DMSO for 10 min, followed by addition of TMAOH to form a biphasic system. After the organic layer was removed, the N3-PMAHcoated QDs were precipitated by isopropyl alcohol and then resuspended in PBS buffer. Unbound N3-PMAH ligands were removed by an ultrafiltration device with a cutoff of 50 kDa. The ultraviolet
visible (UV
vis) absorbance spectra and photoluminescence (PL) spectra of QDs were measured using PerkinElmer Lambda 25 UV
vis absorption spectrophotometer and Edinburgh F900 fluorescent spectrometer, respectively. The morphologies of QDs were observed by TEM using a FEI Tecnai G20 transmission microscope at 200 kV. Dynamic light scattering analysis was performed using a Zetasizer Nano ZS (Malvern Instruments). H5N1p Preparation and Bioorthogonal QD Labeling. H5N1p was prepared in HEK293T cells by transfection with the virus package plasmids as previously described,49 and viral particles in culture supernatants were purified by ultracentrifugation through a 20% sucrose cushion. The concentration of purified H5N1p was determined using a BCA protein assay kit (Pierce). To determine the titer of H5N1p, HEK293T cells were incubated with serially diluted H5N1p solution for 24 h, and the titer of the virus was calculated with the following formula: clone forming units (CFU)/mL = the number of GFP-positive cells/well  dilution folds. For viral particle labeling, purified H5N1p was reacted with 0.1 mM DBCO-PEG4-NHS ester in PBS at 37 °C for 30 min to obtain DBCO-modified H5N1p (DBCO-H5N1p), and unbound DBCO molecules were removed by gel filtration using a NAP-5 desalting column (GE Healthcare). DBCO-H5N1p was then reacted with N3-PMAH-QD750 at different ratios of H5N1p to QDs (w/w) at 37 °C for 1 h to acquire QD-H5N1p.27 In some experiments, H5N1p was heat-inactivated at 65 °C for 10 min, followed by the bioorthogonal QD labeling. The resulting solution was loaded into a 2% (w/v) agarose gel with Trisacetate-EDTA (TAE, pH 8.5) running buffer at 80 V for 80 min and visualized using the Maestro in vivo imaging system (Cambridge Research & Instrumentation). The viral protein in the agarose gel was stained with Coomassie brilliant blue to ensure equal loading of H5N1p. Infectivity and Intracellular Localization of QD-H5N1p in Host Cells. HEK293T cells were infected with H5N1p, DBCO-H5N1p, or QDH5N1p at a multiplicity of infection (MOI) of 0.5 for 48 h, and the infectivity of viral particles was determined by measuring GFP expression using flow cytometry (Becton Dickinson). To investigate the intracellular localization of QD-H5N1p, A549 cells were infected with QD-H5N1p (MOI = 0.5) for 3 to 24 h, followed

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Conflict of Interest: The authors declare no competing financial interest. Acknowledgment. The presented research was financially supported by the National Basic Research Program of China (973 Program No. 2011CB933600), the National Natural Science Foundation of China (81171446, 81071249, 81371679, 20905050), Science and Technology Foundation of Guangdong Province of China (2009A030301010, 2012B090400043, 2012B090600036) and Shenzhen (JC201005270326A, JC201104220242A, JC201005260247A), Guangdong Innovation Research Team of Low-cost Healthcare and SIAT Innovation Program for Excellent Young Researchers (201306). Supporting Information Available: Supplementary methods and results. This material is available free of charge via the Internet at http://pubs.acs.org.

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by immunofluorescent staining with anti-H5N1 hemagglutinin antibodies (Sino Biological Inc.) and Alexa 488-conjugated donkey anti-rabbit IgG secondary antibodies (Life Technology). The fluorescence image of virus-infected cells was recorded using a Nuance FX multispectral imaging system (PerkinElmer). Preparation of Mouse Antiserum against H5N1p. Mouse antiserum was prepared as previously described.49 Briefly, 6 week old female Balb/c mice were subcutaneously immunized by the mixture of H5N1p (20 μg per mouse) in Freund's adjuvant (Sigma) for three times with a 7 day interval, followed by a boost via i.v. injection on day 18. Mouse antiserum containing anti-H5N1p antibodies was collected 4 days after the last immunization, and the titer of anti-H5N1p IgG in the antiserum was 1:128000. Viral Infection Model and In Vivo Imaging. Six week old female Balb/c mice were inoculated intranansally with live or heatinactivated QD-H5N1p (2  108 cfu virus and 20 μg QDs per mouse) in 100 μL of PBS buffer. The equal amount of unconjugated QDs was administered as the negative control. In some mice, QD-H5N1p was pretreated with oseltamivir carboxylate (8 μmol/L) or mouse antiserum against H5N1p (1:1000). Virusinfected mice were monitored by CRi Maestro in vivo imaging system at 12 to 72 h post-infection. At the end of the experiment, images of heart, lung, liver, spleen, and kidney were recorded by the in vivo imaging system, and the average fluorescence intensities were calculated by the following formula: average signals (counts/s/pixel) = total counts/exposure time (s)/area (pixel). Immunofluorescent Staining and Histopathology of Lung Tissues. Mice were sacrificed on day 3 or day 6 post-infections, and lungs were perfused with 4% paraformaldehyde. The fixed lung tissues were cut into 10 μm sections and labeled with antiHA antibodies, followed by Alexa 488-conjugated donkey anti-rabbit IgG. The fluorescent images of QDs and H5N1p in lung tissues were recorded using multispectral fluorescence microscopy. To evaluate H5N1p-induced lung inflammation, lung tissue sections were rehydrated in gradient ethanol solutions (from 100 to 75%), followed by staining with hematoxylin and eosin. Lung pathology was assessed by a previously established histopathological scoring system, and 10
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